The cardiac mitochondria are critical regulators of cell survival during ischemic injury. Considerable research has been directed at interrogating molecular mechanisms that impact their normal and pathological function in the heart. Despite these efforts, to date, very limited information is known regarding the proteomic basis of this vital organelle in the normal and diseased myocardium. Consequently, a fundamental knowledge base to advance our understanding of the role of mitochondria in cardiac disease pathogenesis is lacking. At least two proteomic approaches may be utilized to map the mitochondrial sub-proteome. One focuses on broad-scale annotation of proteins (i.e., expression proteomics) and the second undertakes not only the identification but also the functional characterization of a sub-group of proteins (i.e., functional proteomics). We reason that a simple catalog of proteins without a functional correlate, or a partial catalog of proteins without information regarding the manner in which the mitochondrial proteins are organized to perform their cellular tasks, will be insufficient to generate a thorough understanding of the true proteomic basis of cardiac mitochondrial function. Thus, the application of functional proteomics is more appropriate and critically important to investigate the cardiac mitochondrial sub-proteome, because it gains proteomic information that is linked to the essential biological function of mitochondria. Recent studies have demonstrated that proteins are organized as multiprotein complexes within functional sub- 3roteomes. These multiprotein complexes are assembled to facilitate signal transduction in biological systems. Accordingly, characterization of multiprotein complexes enables not only identification of proteins in sub-proteomes but also characterization of the biological functions that they support. Nevertheless, a versatile functional proteomic Platform specifically designed to target characterization of native mitochondrial multiprotein complexes in the heart is absent. Such a platform would serve as an essential tool to delineate the proteomic basis of mitochondrial function in health and disease, and to decipher the role of multiprotein signaling complexes in this organelle. The central goal of this application is to develop a state-of-the-art proteomic platform with high-sensitivity and high-speed that is tailored for analysis of mitochondrial multiprotein complexes, to gain insights on mitochondrial protein function at a large-scale, and to explore the dynamic modulation of the mitochondrial subproteome during cardiac ischemia. The specific aims are: Aim 1--To develop and optimize a functional proteomic platform to analyze cardiac mitochondrial multiprotein complexes to discern: what proteins make up these complexes, specific protein-protein interactions among proteins within the complexes, and post-translational modifications of these proteins. Aim 2-- To elucidate the effect of myocardial ischemia on mitochondrial multiprotein complexes, protein-protein interactions, protein function, and post-translational modifications using the platform optimized in Aim 1.